The long-term goal of this research is to understand the biochemical mechanism of human amyloid disease, such that novel mechanism-based small molecule and macromolecular therapeutic strategies can be developed. It is now possible to prepare and isolate hybrid transthyretin (TTR) tetramers of defined subunit composition, reflecting those found in heterozygous familial amyloid polyneuropathy patients. In aim 1, the kinetics and thermodynamics of denaturation and amyloidogenicity of the hybrid tetramers will be evaluated as a function of added binding partners and a variety of physiologically relevant conditions to better understand the mechanistic features of amyloidogenesis. The feasibility of a trans-suppression approach for therapeutic intervention will be evaluated, and in related studies, the idea that only certain hybrid tetramers will be amenable to fibril formation at a given denaturation stress level, will be tested. A folded monomeric version of TTR enables the kinetics and thermodynamics of the amyloidogenic tertiary structural changes to be studied independent of the quaternary structural changes. Several hypotheses will be tested, including the idea that amyloid fibrils derived from different sequences have unique quaternary structures. A structure-based design approach for discovering small molecule inhibitors of TTR amyloid fibril formation will be expanded in aim 2 to develop bivalent inhibitors, in an effort to test the amyloid hypothesis in vitro, in cell lines and in a murine animal model in vivo. The kinetics of binding and dissociation of the best structurally diverse inhibitors will be evaluated to better understand their efficacy. In aim 3, the mechanistic connection between amyloidogenesis and the neuropathology characterizing these diseases will be sought. Efficient intralysosomal amyloid formation is observed in a macrophage cell line fed L55P TTR, but not WT TTR. The decreased viability of the cells converting soluble L55P into amyloid allows the use of expression profiling and protein analysis to understand how it is that amyloidosis reprograms the cell for demise. The idea that a rare cell secreting amyloid fibrils (or a lysed amyloid laden cell) could infect cells in its vicinity leading to the rapid onset of disease will be explored also.